Fragment mixtures vs protein mixtures

In FBLD – as in most areas of research – speed and efficiency are prized. The faster you can find quality fragments, the faster you can advance them. NMR-based screening remains one of the most popular fragment-finding methods, and in a recent Molecules paper William Pomerantz and collaborators at the University of Minnesota and Gustavus Adolphus College provide an accelerated workflow.

 

The Pomerantz lab is well known for protein-observed ¹⁹F (PrOF) NMR, in which fluorine-labeled residues are incorporated into proteins. This is easily accomplished by supplementing the media with fluorine-containing amino acids during protein expression. To date more than 15 fluorinated amino acids have been tested in more than 70 proteins, ranging from 7 to 180 kDa in size. Because the chemical shift of fluorine is so sensitive to its environment, a fragment binding nearby can be readily detected by PrOF NMR.

 

When a single type of amino acid is fluorinated, the resulting protein spectrum is considerably simpler than in traditional protein-observed NMR methods. Taking advantage of this, the researchers mixed two different bromodomain proteins: the human oncology target BPTF and PfGCN5 from the malarial parasite Plasmodium falciparum. Both of these bromodomains contain a tryptophan in their N-acetyl-lysine binding sites, so each protein was labeled with 5-flurotryptophan. The proteins were then screened (at 50 µM each) against 467 fragments from Life Chemicals in pools of 4-5 (at 400 µM each). Chemical shift perturbations of the binding-site tryptophan were seen for half of the 98 pools. To determine which fragments were responsible for these shifts, the researchers tested their fragment mixtures against the relevant proteins using (ligand-detected) CPMG NMR. Since they had previously determined the ¹H NMR spectra of all their fragments, it was easy to pick out the binders.

 

Hit rates were similar for both BPTF (9.8%) and PfGCN5 (9.2%), and 4.1% of fragments hit both bromodomains. The researchers had previously screened this library, which is enriched for shapely fragments, against the bromodomain BRD4 D1 (see here) and obtained a similar hit rate. Statistical analyses revealed that the 3D-character for PfGCN5 hits is similar to the library as a whole, as had also been seen for BRD4 D1, while the BPTF hits tended to be flatter.

 

The researchers also followed up on several  fragments individually. One in particular had low micromolar affinity for PfGCN5 as assessed with both PrOF NMR and ¹H-¹⁵N HSQC NMR titrations. Interestingly, this fragment also caused a chemical shift in a different 5-fluorotryptophan residue some 22 Å away from the canonical binding site. Binding at this site could not be competed by a known high-affinity ligand, and a computational screen using FTMap suggested that this does appear to be a secondary binding site.

 

Overall this approach appears to be an appealing workflow as judged by comparing required time, protein, and ligand amounts to other NMR-based screening cascades. As the researchers note, it is advantageous to assess both protein and ligand behavior, as done here. Have you tried using PrOF, and if so how has it performed for you?